Day 1: FACTORS INFLUENCING ENZYME ACTIVITY
Question:
How do factors such as temperature, pH, salinity, and concentration affect the rate of enzyme-facilitated
reaction?
Knowledge Probe:
Guided Inquiry
Enzymes are biological catalysts capable of speeding up chemical reactions by lowering
activation energy. One benefit of enzyme catalysts is that the cell can carry out complex
chemical activities at a relatively low temperature.
Most enzymes are proteins and their 3-dimensional shape is important to their catalytic activity. Two
specific regions on the enzyme structure play an important role in catalytic activity: the active
site and the allosteric site. The active site is the area of the enzyme which binds to the
substance(s) (substrate) and aids in the chemical reaction. The allosteric site is involved in
forming the proper 3-dimensional shape when linked with specific cofactors. As a result of the unique
characteristics of these sites, enzymes are highly specific in terms of the reactions they will catalyze
and the condition under which they work best.
In biochemical reactions the enzyme, combines reversibly with its specific substrate, to form an
enzyme-substrate complex. One result of this temporary union is a reduction in the energy
required to activate the reaction of the substrate molecule so that the products of the reaction, are
formed.
This can be summarized in the equation:
Enzyme + Substrate → Enzyme-Substrate Complex → Enzyme + Product
Note that the enzyme is not consumed in the reaction and can recycle to work with additional substrate
molecules. Each enzyme is specific for a particular reaction because its amino acid sequence is
unique which causes it to have a unique 3-dimensional structure. The active site is the portion of the
enzyme that interacts with the substrate, so that any substance that blocks or changes the shape of the
active site affects the activity of the enzyme.
In practice, this specificity permits one to mix a purified substrate with crude preparations of
enzyme that might contain many other substances and obtain a quantitative assay (analysis) of the
amount of enzyme present.
We will be working in this lab with a representative enzyme —
catalase. Catalase has a molecular weight of approximately
240,000 daltons and contains 4 polypeptide chains, each
composed of more than 500 amino acid monomers. This
enzyme occurs universally in aerobic organisms. One function of
catalase within cells is to prevent the accumulation of toxic levels of
hydrogen peroxide (H2O 2) formed as a by-product of metabolic
processes. Catalase might also take part in some of the many
oxidation reactions going on in all cells.
The primary reaction catalyzed by catalase is the decomposition
of
H2O2
to
form
water
and
oxygen.
2 H2O2 → 2 H2 O + O2 (gas)
In the absence of catalase, this reaction occurs spontaneously, but
very slowly. Catalase speeds up the reaction considerably. In this
experiment, a rate for this reaction will be determined.
The catalase that works in liver
and in red blood cells.
Much can be learned about enzymes by studying the kinetics (changes in rate) of enzymecatalyzed reactions. For example, it is possible to measure the amount of product formed, or the
amount of substrate used, from the moment the reactants are brought together until the reaction
has stopped.
So let’s look at a hypothetical example:
An enzyme and its substrate are mixed in a reaction vessel. If the amount of product formed is
measured at 30 second intervals and this quantity plotted on a graph, a curve like the one in
Figure 1 is obtained:
Figure 1. Enzyme Activity
40
30
Product
(µmoles) 20
10
0
1
2
3
4
5
6
7
8
Time (minutes)
Observe the solid line for this reaction. At time 0 there is no product. After 30 seconds, 5 µmoles
have been formed; after 1 minute, 10; after 2 minutes 20. The rate of reaction could be given as
10 µmoles of product formed per minute for this initial period. Note, however, that by the 3rd and
4th minutes only about 5 additional µmoles of product have been formed. During the first 3
minutes, the rate is constant. From the 3rd minute through the 8th minute, the rate is changing—
it is slowing down. For each successive minute after the first 3 minutes, the amount of product
formed in that interval is less than in the preceding minute. From the 7th minute onward, the
reaction rate is very slow.
In the comparison of kinetics of one reaction with another, a common reference point is needed.
For example, suppose you wanted to compare the effectiveness of catalase obtained from
potato with that of catalase obtained from liver. Would you want to compare the two reactions
during the first few minutes when the rate is constant or later when the rates are changing?
Answer: It is best to compare the reactions when the rates are constant.
In the first few minutes of an enzymatic reaction such as this, the number of substrate
molecules is usually so large compared to the number of enzyme molecules that the enzyme is
constantly having successful collisions with substrate. Therefore, during this early period, the
enzyme is acting on substrate molecules at a constant rate (as fast as it can). The slope of a
graphed line during this early period is called the initial velocity of the reaction. The initial
velocity (or rate) of any enzyme-catalyzed reaction is determined by the characteristics of the
enzyme molecule. It is always the same for a specific enzyme and its substrate as long as
temperature and pH are constant and the substrate is present in excess.
The initial rate of the reaction, therefore is the slope of the linear portion of the curve. To
determine a rate, pick any two points on the straight-line portion of the curve. Divide the
difference in the amount of product formed between these two points by the difference in time
between them. The result will be the rate of the reaction which, if properly calculated, can be
expressed as µmoles of product/second. This equation is:
µmoles2 - µmole1
t2 – t1
In the graph shown as Figure 1:
30 – 20
180 – 120
=
10
60
=
0.17 µmoles/second
The rate of a chemical reaction may be studied in a number of ways, including the following:
1. Measuring the rate of disappearance of substrate, in this example, H2O2
2. Measuring the rate of appearance of product, in this example, O2, which is
given off as a gas.
3. Measuring the heat released (or absorbed) during the reaction.
In this experiment, the disappearance of substrate, H2O2, and the generation of product, O2, are
measured.
Materials:
Obtain the following materials:
•
50mL beaker containing fresh
catalase (yeast) solution
•
hot plate
•
ice
•
reaction chamber
•
thermometer
•
ring stand & clamp
•
boiled catalase
•
10mL graduated cylinder
•
buffers of varying pH: 4, 7, 10
•
100mL graduated cylinder
•
distilled water
•
3% hydrogen peroxide (H2 O2)
•
balance
•
pan (water bath)
•
NaCl (salt)
•
pipette
Investigative Plan:
1. Work as a lab group of 3-4 members. Each lab group will complete Part A and Part B of the
lab. Your teacher will assign on an additional activity to each group form Parts C, D, E & F.
2. At your lab bench you will find a round vial with a rubber stopper top. This is called the
reaction chamber. You will also find a 100mL graduated cylinder, ring stand and clamp,
and a plastic pan which will be used as a water bath. All of this equipment needs to be
assembled into our experimental apparatus, as described and illustrated below.
3. Fill the pan 3/4 full of tap water. Allow the water to come to room temperature.
4. Submerge the 100mL graduated cylinder to fill it with water. Turn the graduated cylinder
upside down, keeping the open end under water, so as to keep it filled with water. Suspend
it upside down in the clamp on the ring stand. Adjust the height of the clamp on the ring
stand so the open end of the graduated cylinder is about 3cm above the bottom of the pan.
See diagram below.
5. Place a thermometer in the pan and record the temperature
of the water, during Part A of the lab.
6. When all sections of the lab are complete, share the data with the class from your group’s
section. Each person must plot the data for all parts of the lab on his/her own graph paper.
°C
FACTORS INFLUENCING THE ACTIVITY OF CATALASE
Several enzymatic variables will be examined in this lab. You will be using the protein enzyme,
catalase. Catalase is found in most cells, even in single-celled eukaryotes like yeast. In this lab,
you will extract catalase from a yeast solution and test its catalytic effectiveness on hydrogen
peroxide. Catalase speeds up the breakdown of peroxides which may form during respiration
(metabolic energy production). This breakdown prevents the peroxide from causing unwanted
oxidation of important biomolecules:
2 H2O2 → 2 H2O + O2 (gas)
We will measure enzyme activity by measuring the generation of oxygen gas — a
product in the reaction.
Part A. The Time Course of Enzyme Activity
1. Set up the experimental apparatus as illustrated and described above.
2. Obtain a reaction chamber.
3. Obtain a bottle of 3% hydrogen peroxide (H2O2) solution and a 10mL graduated cylinder.
4. Obtain a small amount of stock catalase (yeast) solution in a 50mL beaker. You will
need 1.0mL of yeast solution for each trial. When you are ready, you will add it to the vial
with a plastic pipette.
5. Pour 10mL of hydrogen peroxide (H2O2) into the reaction chamber. Pipette in 1.0mL of
stock catalase solution (yeast solution) and IMMEDIATELY stopper the reaction
chamber tightly, submerge it in the water bath and place the plastic tubing into the
bottom of the graduated cylinder, so all the bubbles formed in the reaction chamber are
captured by the inverted graduated cylinder.
6. Measure the gas levels in the graduated cylinder at 30-second intervals for 5 minutes.
(In the observation section): Record the levels in a data table of your own design.
7. (In the data analysis section): Plot the data on a graph. Don’t forget to label your axes
and title your graph.
Part B. The Effect of Enzyme Concentration on Enzyme Activity
1. Repeat the experiment from Part A, using 3 different levels of enzyme concentration:
75%, 50%, and 25% concentration of enzyme solution. You may easily do this by using
the following procedures:
a. 75% concentration: Follow the procedure from Part A, but use 0.75mL catalase
solution in the reaction chamber, instead of 1.0mL.
b. 50% concentration: Follow the procedure from Part A, but use 0.50mL catalase
solution in the reaction chamber, instead of 1.0mL.
c. 25% concentration: Follow the procedure from Part A, but use 0.25mL catalase
solution in the reaction chamber, instead of 1.0mL.
2. (In the observation section): Record all data in a data table of your own design.
3. (In the data analysis section): Plot the data on the same graph as Part A. Don’t
forget to clearly label the enzyme concentrations on your plotted lines.
Day 2: FACTORS INFLUENCING ENZYME ACTIVITY
Part C. The Effect of Temperature on Enzyme Activity
Design how to test the effects of temperature on enzyme activity. How many trials will you need? What
temperatures will you use and why? Write out your investigative plan.
Part D. The Effect of pH on Enzyme Activity
Design how to test the effects of pH on enzyme activity. How many trials will you need? What pH levels
will you use and why? Write out your investigative plan.
Part E. The Effect of Substrate Concentration on Enzyme Activity
Design how to test the effects of substrate concentration on enzyme activity. How many trials will you
need? What enzyme concentrations will you use and why? Write out your investigative plan.
Part F. The Effect of Ionic Concentration on Enzyme Activity
Here you will use different concentrations of salt solution. Your strongest solution will be 10% NaCl.
Design how to test the effects of ionic concentrations on enzyme activity. How many trials will you
need? What ionic concentrations will you use and why? Write out your investigative plan.
Observation:
Data Analysis:
Explanation:
→Claim-- Does the claim answer the investigation question?
→Evidence-- Does the selected evidence support the claim?
→Reasoning-- Does the reasoning link the claim and evidence?
Does the reasoning justify why the evidence supports the claim?
Does the reasoning make a strong argument?
Does the reasoning consider alternative explanations?
(Use one group as an example first)--Have partner groups present and defend to each other and then
come up with a recommendation for the whole class.
Evaluation—
a.
b.
c.
d.
e.
f.
What are the sources of error?
What would you do differently next time?
How confident are you in your results? (See Confidence Chart) What could be alternative
explanations for my results?
What surprised you?
What would your prediction be if you conducted this investigation again?
What question would you like to pursue next?
→Application: How do I use what I learned?